【作者】 冯浩；

【导师】 尹时华；

【作者基本信息】 广西医科大学 ， 耳鼻咽喉头颈外科学， 2010， 硕士

【摘要】 目的研究水杨酸钠（sodium salicylate, SS）诱导豚鼠耳蜗螺旋神经节细胞（spiral ganglion neuron, SGN）凋亡及凋亡的机制。方法40只成年花色雄性黑目豚鼠随机分为4组,每组10只。A组:空白对照组;B组:人工外淋巴液（artificial perilymph, APL）组（左耳经圆窗龛注入APL后未给SS）;C组:SS(400 mg·kg^-1·d^-1)+APL组（左耳经圆窗龛注入APL后经腹腔SS给药）;D组:SS(400 mg·kg^-1·d^-1)+zDEVD-FMK组（左耳经圆窗龛注入zDEVD-FMK后经腹腔SS给药）。所有动物均于给药前后分别进行听性脑干反应（auditory brainstem response, ABR）阈值测试。10 % SS（溶于APL配制而成）均采用腹腔注射给药方式,400 mg·kg^-1·d^-1,并于连续给药10天后即时处死动物取出耳蜗,采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling, TUNEL）检测耳蜗SGN凋亡细胞,免疫组织化学染色方法检测SGN内激活型caspase-3蛋白表达情况,透射电镜（transmission electron microscopy, TEM）观察SGN超微结构变化。结果ABR测试结果显示A、B两组动物听阈均无明显变化,C组动物给药后听阈提高,与A、B两组比较有显著性差异（p<0.01,p<0.01）,D组动物听阈较C组降低（p<0.05）;TUNEL标记显示A、B两组动物耳蜗中均未见SGN凋亡细胞,亦无激活型caspase-3蛋白表达,而C组则出现较多TUNEL阳性着色SGN,并伴有激活型caspase-3表达的增高,D组较C组TUNEL阳性着色SGN减少,激活型caspase-3表达亦降低,A、B两组与C、D两组比较差异均有显著统计学意义（p<0.01,p<0.01）,而C、D两组比较亦有显著差别（p<0.01）;透射电镜观察结果显示A、B两组SGN超微结构正常,C组SGN伴有明显细胞凋亡形态学特征,D组SGN超微结构未见明显凋亡征象。结论①SS可诱导豚鼠耳蜗SGN发生凋亡;②SGN凋亡与caspase-3激活有关;③zDEVD-FMK可在一定程度上拮抗caspase-3的激活,并阻断其引起的SGN凋亡。更多还原

【Abstract】 Objective: In the present study, in vivo experiments are conducted to test efficacy of caspase-3 inhibitor zDEVD-FMK for protecting spiral ganglion neuron （SGN） from salicylate-induced apoptosis in the guinea pig cochlea.Methods: Forty adult guinea pigs were divided randomly into four experimental groups, each for ten, they were: Group A, served as blank control without any disposal; Group B, APL only, The animals were disposed with APL through RWN by the means of cochlear micro-injection, the left ear was implemented with an operation; Group C, APL plus salicylate. The animals were treated with APL through RWN before drug injection, with a does of 400 mg·kg^-1·d^-1 i.p., for ten consecutive days; Group D, zDEVD-FMK plus sodium salicylate. The animals were treated with caspase-3 inhibitor zDEVD-FMK through RWN before drug exposure in the same way described in the Group C. All animals received ABR thresholds measurement before drug administration and after last injection; they were then sacrificed for cochleae followed by staining. Programmed cell death （PCD） executioner was evaluated with immunohistochemistry detection of activated caspase-3. Apoptosis was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling （TUNEL） method. Transmission electron microscopy （TEM） was also used to evaluate morphological change in the auditory neuron.Results: Animals disposed with salicylate appeared a high elevation in response of ABR thresholds testing （p<0.01 vs. Group A and B）. By contrast, for those received zDEVD-FMK in advance, their hearing improved （p<0.05 vs. Group C）. Salicylate strengthened immune reaction in the fields of SGNs, which was in accordance with apoptotic auditory effectors emergence （p<0.01 vs. Group A and B）. On the contrary, topical inhibitor delivery alleviated pigmentation of nuclei （p<0.01 vs. Group C）, keeping pace with antigen expression decrease, but a few immunostaining could be still detected （p<0.01 vs. Group C）. With TEM to SGNs, no ultrastructural change occurred in the Group A and B, while neurons obtained from Group C demonstrated obvious apoptotic changes, characterized with chromatin condensation and nucleus margination. Treatment with zDEVD-FMK prevented the salicylate-damaged SGNs by apoptosis in a highly significant manner.Conclusion: These findings suggest that long-term administration of high-dose salicylate can activate caspase-3 pathway to induce SGN apoptosis.更多还原